Electrophoresis is a well known analytical technique in biochemistry. A sample is placed in a matrix and exposed to an electric field which causes various components in the sample to migrate within the matrix at different rates depending on the component's charge, molecular weight and other physical and chemical properties. After migration has occurred, the resulting migration pattern is ascertained. Various methods to ascertain the migration pattern have been developed. These include autoradiography and staining for visual or densitomeric determination. Typical stains include the dyes Coomassie Brilliant Blue and Ponceau S. Silver staining has been used to increase sensitivity over that provided by dyes. A widely used silver staining technique is that described by Merril et al., Methods in Enzymology, Volume 96, p. 230 (1983). An electrophoresis matrix, specifically polyacrylamide, is immersed in either an acid or an acid/alcohol solution for about one hour to fix the protein in the matrix. The matrix is then washed, typically for thirty minutes. The matrix is then soaked for about five minutes in a dichromic acid solution to oxidize the protein. Next, the gels are soaked in a silver nitrate solution for twenty minutes and then rinsed with a sodium carbonate/formaldehyde buffer to reduce silver ion bound to proteins and nucleic acids. A silver pattern is then allowed to develop. Development is stopped with acetic acid. The pattern is then analyzed either by direct visualization or by instrumental techniques.
The method of Merril et al. was simplified by Oakley et al. ([Analytical Biochem., Volume 105, p. 361 (1980)]. Electrophoresis gels were treated with unbuffered glutaraldehyde to cross-link proteins. Following rinsing, the gels were treated with ammoniacal silver solution. A combination of citric acid and formaldehyde was used to reduce silver ion to silver.
It has been found that the sensitivity of the silver staining technique for the optical detection of proteins and nucleic acids can be improved substantially if the matrix is treated with a fixing agent comprising a highly aromatic compound having at least one sulfonic acid group and at least one aromatic, tertiary amine, preferably as part of an oxazole group. Preferred compounds are selected from the group consisting of ##STR1## wherein R is H, CH.sub.3, C.sub.2 H.sub.5 or CH.sub.2 N.sup.+ (CH.sub.3).sub.3, ##STR2## Optionally, the matrix is treated with a sensitizing agent selected from the group consisting of sodium sulfide, dithiothreitol, thiourea and sodium thiosulfate.
In addition, the sensitivity of the silver staining technique for the optical detection of nucleic acids can be improved substantially if the matrix is treated with a fixing agent comprising a compound of the formula: ##STR3##
The increase in sensitivity for both protein and nucleic acids is believed to result from the ability of these fixing agents to cross-link proteins and nucleic acids while, at the same time, providing an aromatic ring containing a tertiary amine which is capable of forming a coordination complex with silver.